This invention relates to polypeptide hormone analogues that exhibit enhanced pharmaceutical properties, such as increased thermodynamic stability, decreased mitogenicity, and feasibility of a rapid-acting formulation at high protein concentrations (1-5 mM) in the absence of zinc ions. More particularly, this invention relates to insulin analogues that confer altered or selective post-receptor signaling properties (relative to signaling by wild-type insulin). The insulin analogues of the present invention thus consist of two polypeptide chains that contain a novel combination of amino-acid substitutions such that the analogues exhibit (i) enhanced thermodynamic stability, (ii) decreased self-association at protein concentrations greater than 0.6 mM, and (iii) at least a portion of the biological potency of the human insulin molecule, although a greater number of protein molecules may be required, on subcutaneous or intravenous injection in a mammal, to elicit a similar reduction in blood-glucose concentration.
The engineering of non-standard proteins, including therapeutic agents and vaccines, may have broad medical and societal benefits. Naturally occurring proteinsas encoded in the genomes of human beings, other mammals, vertebrate organisms, invertebrate organisms, or eukaryotic cells in general—may have evolved to function optimally within a cellular context but may be suboptimal for therapeutic applications. Analogues of such proteins may exhibit improved biophysical, biochemical, or biological properties. A benefit of protein analogues would be to achieve enhanced “on-target” activity (such as metabolic regulation of metabolism leading to reduction in blood-glucose concentration) with decreased unintended and unfavorable side effects, such as promotion of the growth of cancer cells or increased biosynthesis of lipids. Another benefit of such protein engineering would be preservation of rapid onset of action on concentration of the protein to achieve formulations of higher strength. Yet another example of a societal benefit would be augmented resistance to degradation at or above room temperature, facilitating transport, distribution, and use. An example of a therapeutic protein is provided by insulin. Wild-type human insulin and insulin molecules encoded in the genomes of other mammals bind to insulin receptors is multiple organs and diverse types of cells, irrespective of the receptor isoform generated by alternative modes of RNA splicing or by alternative patterns of post-translational glycosylation. Wild-type insulin also binds with lower but significant affinity to the homologous Type 1 insulin-like growth factor receptor (IGF-1R).
Insulin is a two-chain protein molecule that in a vertebrate animal is the biosynthetic product of a single-chain precursor, designated proinsulin. The sequence and structure of human proinsulin are illustrated in FIGS. 1A and 1B, respectively; the sequence of human insulin is shown in FIG. 1C. The two polypeptide chains of insulin are respectively designated A and B. Specific residues in one or the other chain are designated below by standard three letter code (for example, Ala for Alanine or Asp for Aspartic Acid) followed by a superscript that designates the chain (A or B) and residue number in that chain. For example, Histidine at position 10 of the B chain is designated HisB10, Valine at position 12 of the B chain is designated ValB12, and Threonine at position 8 of the A chain is designated ThrA8. “Insulin analogues” designate a class of molecules related to wild-type insulin by substitution of one more amino-acid residues by a different type of amino acid or by modifications of one or more atoms in the side chain or main chain of such residues by a different atom or set of atoms. An example of an insulin analogue known in the art is insulin lispro, in which ProB28 is substituted by Lys and LysB29 is substituted by Pro. Insulin lispro (also designated KP-insulin) is the active component of the product Humalog® (Eli Lilly and Co.).
It is known in the art that the B chain of insulin may be modified through standard amino-acid substitutions at one or a few positions to enhance the rate of absorption of an insulin analogue formulation from the subcutaneous depot. An example of a further medical benefit would be optimization of the pharmacokinetic properties of a soluble insulin analogue formulation such that rapid onset of action is retained in formulations of strengths in the range U-200 through U-1000, i.e., between twofold and tenfold higher than conventional U-100 insulin products (in this nomenclature “U-X” designates X internal units per ml of solution or suspension). Insulin formulations of increased strength promise to be of particular benefit for patients who exhibit marked insulin resistance and may also be of value in internal or external insulin pumps, either to extend the reservoir life or to permit miniaturization of the reservoir in a new generation of pump technologies. Existing insulin products typically exhibit prolonged pharmacokinetic and pharmacodynamics properties on increasing the concentration of the insulin or insulin analogue to achieve formulation strengths>U-200 (200 international units/ml). Such prolongation impairs the efficacy of such products for the prandial control of glycemia on subcutaneous injection and impairs the efficacy and safety of pump-based continuous subcutaneous infusion. In light of these disadvantages, the therapeutic and societal benefits of rapid-acting insulin analogue formulations would be enhanced by the engineering of insulin analogues that retain rapid action at strengths between U-200 and U-1000. Additional benefits would accrue if the novel soluble insulin analogue exhibited weaker affinity for the Type 1 IGF receptor relative to wild-type human insulin. Still additional therapeutic and societal benefit would accrue if the concentrated insulin analogue formulation should exhibit reduced mitogenicity in assays developed to monitor insulin-stimulated proliferation of human cancer cell lines.
Administration of insulin has long been established as a treatment for diabetes mellitus. A major goal of conventional insulin replacement therapy in patients with diabetes mellitus is tight control of the blood glucose concentration to prevent its excursion above or below the normal range characteristic of healthy human subjects. Excursions below the normal range are associated with immediate adrenergic or neuroglycopenic symptoms, which in severe episodes lead to convulsions, coma, and death. Excursions above the normal range are associated with increased long-term risk of microvascular disease, including retinapathy, blindness, and renal failure. Although the importance of glycemic control is well known in the art, the pathophysiology of type 2 diabetes mellitus (T2DM) is also characterized by selective insulin resistance (SIR) where insulin becomes ineffective at glycemic control and yet continues to drive mitogenicity and excess lipid synthesis. Accumulating lipid in the liver and muscle further unbalances glucose regulation, increases insulin resistance, and accelerates the progression of T2DM and its complications. To our knowledge, there are presently no insulin products (approved or in clinical trials) that rebalance such perturbed cellular and organ-specific signaling. We thus anticipate that such a product would create a new treatment paradigm in T2DM, yielding significant long-term health benefits and reduction in aggregate health-care costs.
Insulin is a small globular protein that plays a central role in metabolism in vertebrates. Insulin contains two chains, an A chain, containing 21 residues, and a B chain containing 30 residues. The hormone is stored in the pancreatic β-cell as a Zn2+-stabilized hexamer, but functions as a Zn2+-free monomer in the bloodstream. Insulin is the product of a single-chain precursor, proinsulin, in which a connecting region (35 residues) links the C-terminal residue of B chain (residue B30) to the N-terminal residue of the A chain. A variety of evidence indicates that it consists of an insulin-like core and disordered connecting peptide. Formation of three specific disulfide bridges (A6-A11, A7-B7, and A20-B19) is coupled to oxidative folding of proinsulin in the rough endoplasmic reticulum (ER). Proinsulin is coverted to insulin in the trans-Golgi network en route to storage as zinc insulin hexamers in the glucose-regulated secretory granules within pancreatic beta-cells. The classical crystal structure of insulin (one protomer extracted from the zinc hexamer) is shown in FIG. 2.
The present invention was motivated by medical and societal needs to engineer a rapid-acting insulin analogue in a soluble formulation at neutral pH at strengths in the range U-100 through U-1000 that exhibits altered or selective post-receptor signaling properties. A barrier to such products has long been posed by the prevailing paradigm of how binding to the insulin receptor leads to transmission of a signal across the cellular membrane, leading to autophosphorylation of the cytoplasmic portion of the receptor. Such autophosphorylation in turn activates a variety of post-receptor signaling pathways, such as pathways leading to (i) translocation of the GLUT4 glucose transporter from an intracellular compartment to the plasma membrane, (ii) transcriptional activation of genes promoting the growth and proliferation of cancer cells, (iii) storage of glucose molecules within the cell as glycogen, and (iv) metabolic transformation of the insulin molecule through the intracellular biosynthesis of lipids.
It is not known in general whether or how the insulin molecule may be modified such that one post-receptor signaling pathway may be selectively strengthened or attenuated. The structure of the intact insulin receptor has not to date been determined, and so the mechanism of how binding of insulin to the outside of the cell (the “ectodomain” of the receptor) of the receptor) leads to propagation of a signal to the inside of the cell (i.e., to the cytoplasmic domain of the receptor) is not known. A crystal structure of the apo-ectodomain is known in the art as an inverted-V dimeric assembly at low resolution (FIG. 3) but crystals could not obtained with an insulin molecule bound. The crystal structure of insulin bound to a domain-minimized “micro-receptor” has also been determined at low resolution (FIG. 4), but this construction lacks the beta-subunit of the receptor required for trans-membrane signaling and communication of a signal to post-receptor pathways. Accordingly, it is not known in the art whether or how modification of the insulin molecule might affect the relative strength of various post-receptor signaling outputs.
An insulin analogue known in the art to exhibit an unfavorable change in the balance of post-receptor signaling is provided by AspB10-insulin. The original motivation for the design and preparation of this analogue was based on its structural role in insulin self-assembly. The wild-type residue (HisB10) functions in native hexamer assembly to coordinate the two axial zinc ions in the central axis of the hexamer. Substitution of HisB10 by Asp impairs the binding of zinc ions in this axial mode and blocks higher-order self-assembly via the trimer-related surface of the classical hexamer. AspB10 may be expected on general grounds by enhance the segmental stability of the central B-chain α-helix in the zinc-free monomer or dimer via electrostatic mechanisms: as a favorable C-Cap residue and through potential formation of an (i, i+4) salt bridge. Irrespective of the theoretical underpinnings of protein stability, substitution of HisB10 by Asp was observed indeed to augment the thermodynamic stability of the zinc-free insulin monomer as probed by chemical-denaturation studies. AspB10 also enhances the affinity of insulin for the insulin receptor and augments in parallel its potency to stimulate lipogenesis in isolated adipocytes.
Despite the above favorable structural and biophysical properties conferred by substitution of HisB10 by Asp in wild-type insulin, its clinical use was precluded by increased mitogenicity in cell-culture assays of neoplastic cell lines (including a cell line derived from a human breast cancer) in association with the finding of an excess incidence of mammary tumors on chronic treatment of Sprague-Dawley rats by AspB10-insulin relative to wild-type insulin. The present invention provides a combination of a non-standard amino-acid substitution in the insulin molecule (penta-fluoro-PheB24) such that the favorable properties conferred by AspB10 (such as enhanced stability and impaired self-assembly beyond the stage of dimerization) are retained whereas the unfavorable increase in mitogenicity in cell-culture assay is mitigated or even reserved to achieve a level of mitogenicity lower than that of wild-type insulin itself. As a further surprise, the combination of AspB10 with penta-fluoro-PheB12 favorable alters the balance of post-receptor signaling pathways in muscle such that formation of glycogen is enhanced relative to formation of lipids.